DESCRIPTION. (Adapted from Applicant's Abstract) Cryptococcus neoformans produces large amounts of mannitol in culture and in infected animals, and a mannitol-underproducing C. neoformans mutant is stress-intolerant and hypovirulent. Therefore, the PI will examine the hypothesis that C. neoforman's abilities to synthesize mannitol and to regulate its intracellular mannitol concentrations are required for wild-type stress tolerance and virulence. Aim 1 is to elucidate the mannitol biosynthetic pathway in C. neoformans and to ascertain this pathway's functional and pathogenic significance. In other fungi, mannitol-1-phosphate dehydrogenase (MPD) catalyzes a key step in mannitol biosynthesis. The PI has purified C. neoformans MPD to homogeneity and cloned its cDNA. Therefore, he will (i) clone and sequence the C. neoformans MPD structural gene (MPD1), (ii) construct mpd1 null mutants, and (iii) test these mutants for their abilities to synthesize and catabolize mannitol, tolerate environmental stress, and cause disease in animals. Aim 2 is to elucidate the mannitol catabolic pathway in C. neoformans and to ascertain this pathway's functional and pathogenic significance. In other fungi, NAD- or NADP-linked mannitol dehydrogenases (MDHs) catalyze key steps in mannitol catabolism, and the PI has demonstrated an NAD-linked MDH (NAD-MDH) in C. neoformans. Therefore, he will (i) clone the C. neoformans NAD-MDH gene (MDH1), (ii) construct mdh1 null mutants, and (iii) test these mutants for their abilities to synthesize and catabolize mannitol, tolerate environmental stresses, and cause disease in animals. Aim 3 is to examine the roles of MPD and NAD-MDH in regulating the biosynthesis, catabolism and intracellular accumulation of mannitol in C. neoformans. Total and intracellular mannitol produced in various environmental conditions will be measured and correlated with MPD and NAD-MDH mRNA and protein levels. Also, the functions of a C. neoformans regulatory gene (MTL1) that permits Saccharomyces cerevisiae to express a cryptic NAD-MDH will be studied by overexpressing MTL1 in C. neoformans.